Medicinal compound



in a. water-soluble form.

Patented Mar. 23, 1937 2,074,492 MEDICINAL COMPOUND Michigan No Drawing.

Serial No. 478,558-

7 Claims;

The invention relates to an extract obtainable from mammalian suprarenalglands and a method of preparing the same. invention pertains to anextract from the suprarenal cortex containing in concentrated form theactive principle or hormone of the adrenal cortex.

Another object of our invention is to provide an improved process fortreating the cortex of mammalian suprarenal glands whereby the activeprinciple or hormone may be obtained in a concentrated form useful foranimal and human therapy in cases of deficient functioning of theadrenal cortex.

A further object is to obtain a practically pure extract of adrenalcortex, free from injurious substances including inert associated glandtis-, sue, proteins, salts, lipoids and substantially free of adrenalin.

These and other objects are attained by treating mammalian suprarenalglands in the manner hereinafter set forth.

In general our improved process consists in subjecting fresh mammaliansuprarenal cortex to a series of extractions by organic reagents soselected as to successively remove from the starting material thevarious substances associated with the hormone, finally leaving aproduct'containing the active principle in a purified state and in highconcentration.

We have found that the active principle or hormone of the suprarenalcortex can be ex tracted from the gland material by means of lipoidsolvents such as benzol, sulphuric ether, petroleum ether, etc., but theactive principle itself is not a fat andmay be separated from fats andcholesterol-like compounds and be obtained The use of a lipoid solventfor the extraction of the hormone has the advantagethat proteins andadrenalin are readily removed.

In the extraction of the product we prefer to apply first an organicsolvent which is also soluble for example ethyl alcohol, propyl alcohol,acetone, etc., and then to subject the extracted material after removalof the watersoluble solvent to a more typical lipoid solvent such asbenzol, petroleum ether, carbon tetra More particularly the' chloride,etc.

like benzol but on the sequence of tw an organic solvent Under propertions the comminuted gland tracted directly with Wilbur Willis Swingleand Joseph John Pfiffner,

Princeton,' N. J., assignors Company, Detroit, Mich,

to Parke, Davis 80 a. corporation of Application August 28', 193

a laboratory s PATENT OFFICE mechanical conditissue can be exawater-insoluble solvent scale we prefer tractive agents, one beingsoluble in water and the second an organic solvent insoluble Th lipoidfractions thus obtained contain the active'principle necessary ofsuprarenalectomized animals rtain disadvantages due to the but theypossess ce presence of lipoidal im however, that the phospholi in water.

for maintaining the life (cats or dogs) purities. We have found,

pids can be separated in the recognized way, using acetone, without lossof the active principle.

We have been able also to separate the hormone from fats and cholesterolby an elaborate system of selective solvents, such as aqueous alcohol.

distribution between suitable between petroleum ether and The major partof the cholesterol and neutral-fat is then found in the hydrocarbonsolvent and th principle or We have hormone i demonstrated e major partof the active 11 the alcohol-water layer. that the active principle iscomparatively stable and not readily destroyed under the conditions thatwe have devised.

While our process out in various speci general steps outlined in makinga very poten following process.

from the animals immedi should of the process For convenience, thedirec- The adrenal glands from c tions hereinafter given are based upona lot of adrenal cortex weighing 2800 3500 grams.

attle are obtained ately after slaughter and til ready for processingwhich thin twenty-four d extraneous fat are removed, the glands splitlengthwise, and the 'medullary tissue scraped out. The cortical tissueis then passed through an ordinary using the finest cutter.

Preparation of crude alcoholic extract The ground cortical tissue isplacedinto an earthenware ar alcohol added.

temperature with to six days, after muslin and the re press. The softpaper.

d 2 volumes of 95% meat grinder ethyl stand at room occasional stirringfor three which it is strained through sidue expresse liquid product isthen The gland residue is d in a tincture filtered through reground andextracted in a similar manner for three days with 2 volumes of 80% ethylalcohol.

The alcoholic t extract by means of the about 500 cc.) is

more.

external After decanting the residue is transferred to a mortar andrubbed with five 100 cc...portions of fresh cold acetone.

mixed. The 70% 75 fairly high speed for 30 minutes.

extracts are concentrated separately in partial vacuo at. externaltemperature -60'? C. to about 1/15 their original volumes.

5 Removal of proteins, much inert material, and

. most of the adrenalin From this stage on all material is kept in therefrigerator except when under Each aqueous residue obtained above(volume transferred at once to a liter to settle. The benzene solutionis removed by and the aqueous residue washed twice The last benzenewashing is colorless or only slightly yellow. The benzene washings areall combined. Preferably a large centrifuge is used to separate thebenzene extract. The benzene is completely removed in partial vacuo attemperature 45-50 C. using atwo liter balloon flask and allowing thematerial to enter the flask as rapidly as the benzene. distils. Thisbenzene can be used repeatedly for the same pur- 25 pose without anypurification.

Removal of phospholipids To the residue in theflask is added 500 cc.acetone. The material is rubbed thoroughly with a heavy bent glass rodand set aside in the refriger- 3 atorfor twenty-four hours withoccasional rubbing. The acetone solution is then decanted and theresidue reextracted in the same manner. the second acetone extractivesThe acetone extractives are combined, filtered and the acetone removedin partial vacuo at 45 50 C. external temperature.

Removal of cholesterol and neatral fat The residue is transferred to aseparatory funnel with 30 cc. petroleum ether (13. P. 30.or 40- and 74cc. 95% ethyl alcohol. 26 cc. distilled water are added and the contentsgently alcoholic solution which separates is washed 5 times with 30petroleum ether.

and the petroleum ether removed in partial vacuo 0 at externaltemperature 40-50 C.v The above procedure is repeated. The petroleumether solution resulting from thesecond distribution is run into anotherfunnel, 74 cc. of 95% alcohol added and then 26 cc. distilled water. Thepe- 55 troleum ether solution resulting from the third run into anotherfunnel 74 cc. of

distribution is 95% alcohol added and then 26 cc. distilled water. Thisgives a total of ,4 distributions, The alcoholic solutions are washedsuccessively 5 times the 3rd and 4th distributions are then washed twicewith fresh 30 cc. portions manipulation.

Distillation is continued to a vol-' carefully decanted from the blackoily appearing material which is thrown down and passed through a largeSeitz filter.

The extract of suprarenal cortex therapeutically without anyobjectionable reaction.

The physiological properties of the extract are 'best shown by testswhich have been madeon laboratory animals such as bilaterallyadrenalectomized cats. It is a well established fact that the life of acat after suchoperation is seldom more than 8 to 10 days and heretoforeit has been impossible to extend the life of such animals for anyappreciable span by the administration of any known substance, or by anyknown treatment. However, the daily subcutaneous administration of ourextract in amounts from '0.5 to 1.0 cc. per kilogram of body weight hasresulted in maintaining the life span for 100 days or more followingadrenalectomizing in every instance, and'there has been no indicationthat this life span might not be prolonged indefinitely by the sametreatment. on the other hand, cats which appeared normal during theadministration of our extract showed the characteristic symptoms ofadrenal insumciency as soon as the administration of the extract wasdiscontinued, and. death always resulted'within 8 to 10 days of thistime. Therefore it has been definitely shown that our extract hasphysiological properties correspondingto the hormone of the suprarenalcorte Our extract has also been used in human therapy in cases wheredeficiency in suprarenal cortex was indicated and it has had beneficialeffects in such cases.

- What we claim as our invention is:

1. A process for obtaining an active extract from the cortical portionof the suprarenal glands in concentrated form and sufiiciently free fromimpurities to have the physiological characteristic of alleviatingsymptoms of adrenal insufficiency which comprises extraction of freshsuprarenal cortex with an alcohol capable of dissolving the activesubstance without destroying the activity thereof, separating thesolvent from the solid matter, removal of the alcoholsolvent, extractionof the resulting residue with a liquid hydrocarbon of low molecularweight capable of dissolving the active substance without destroying thesame and incapable of dissolving substantial amounts of proteins, saltsor adrenalin, separation of the so-- lution from the solid matter,removal of said hydrocarbon solvent, extraction of the residue withacetone capable of dissolving the active substance respectively capableof separating by distribution the active substance from portions ofneutral-fat, adrenalin and other impurities, removal of the aqueousalcohol containing the active principle from the petroleum ether,removing the alcohol leaving an aqueous solution.

separation of the aqueous solution from solid precipitate after each ofsaidsolvent treatments,

matter, and sterilization of said'solution. removing the solvent aftereach of said separa- 4 2. A process for obtaining an active extracttions, and subjecting the residue remaining after from the corticalportion of the suprarenal glands the removal of said third solvent to afractional 5 in concentrated form and sufliciently free fromdistribution between aqueous alcohol and, petro- 5 impurities to havethe physiological character--. le'umether, thereby obtaining thecortical horistic of alleviating symptoms oi adrenal insufllmone in theaqueous alcohol. ciency which comprises extraction of fresh su- 5. Theprocess for obtainin an extract of the prarenal cortex with an alcoholcapable of discortical hormone from -he suprarenalglands solving theactive substance without destroying comprising treating suprarenalcortex tissue with 10 I the activity thereof, separating the solventfrom alcohol, separating the solution from the solid the solid matter,removal of the alcohol solvent, matter, removing the alcohol from thesolution, extraction of the'resulting solution with benzene treating theproduct after alcohol removal with capable of dissolving the activesubstance without benzene, separating the solution from the soliddestroying the same and incapable of dissolving matter, removing thebenzene from said solution, substantial amounts of proteins, salts oradren treating the product after benzene removal with alin, separationofthesolution from thesolid acetone, separating the solution .from thesolid 'matter, removal of said benzene solvent, extracmatter,. re movingthe acetone from said solution "tion of the residue with acetonecapableof disand subjecting'the product remaining after said solving theactive substance and preserving the acetone removal to repeateddistributions be;- 20

activity thereof and incapable of dissolving subtween aqueous alcoholand petroleum ether, comstantial portions of phospholiplds, removal ofthe bining the aqueous alcohol fractions, removing v solution from thesolid matter, removal} of said the alcohol leaving an aqueous solutionand sepaacetone solvent, treatment of the residue withrating the aqueoussolution from the solid matter.,

twofimmiscible reagents capable of separating I 6. An'extractof-thesuprarenal cortex derived 25 by distribution the active substance"from p'orfrom the cortical portion of the suprarenal glands, 'tions ofneutralfat, adrenalin and other impurisaid extract being a clearpale-yellow solution ties, said reagents'com'prising petroleum ether oncontaining the cortical'hormone sufiiciently- 'free the one hand and amixture of alcohol and water from epinephrine and other impurities to besui t on the other handfiremovah ofthe alcohol and able forsubcutaneous, intraperitoneal or intra- 30 water reagent containing theactive principle from venous injection of human beings suffering, fromthe other immiscible reagent, removal of the al- .Addisons disease insuflicient quantities to comcohol leaving an aqueous solution containingthe bat adrenal cortex insufliciency, said extract be active substance,separation of the solution from ing sufficiently potent to permitadrenalcortex resolid matter and terilization of said solution. a placement.therapy in bilaterally .adrenalecto- 3'5 3. The proces for obtaining anextract of the mized'animals and to maintain the lives of said corticalhormone from the; suprarenal glands animals for over 100 days followingadrenaleccomprising treating suprarenal' cortex tissue; with tomy'and.to restore to normal conditionsanimals an organic lipoid solvent of theclass consisting prostrated by adrenal cortex deficiency.

40 of benzol, sulphuric ether, petroleum ether and j 1. An extractofthesuprarenal cortex derived 40 carbon tetrachloride, removing saidsolvent, leav-.- from the cortical portion of the suprarenal glands, inga residue,'treating said residue with acetone said extract when insolutionform being a clear to precipitate lipoids, removing said acetoneleavsolution containing the cortical hormone js ufiiing a residue andsubjecting thelast mentioned ciently free irom epinephrineandotherimpuri l5 residue to a fractional distribution between aque- .ties to besuitable for subcutaneous, intraperious alcohol and petroleumether,therebyobtaintoneal .or intravenous injection of human beings ing thecortical hormone in the aqueous alcohol. suffering fiom Addison's.disease in sufficient 4. The process for obtaining an extract. of the,quantities to combat adrenal cortex insufilciency, corfcal hormone fromthe suprarenal glands said extract being suificiently potent to permitcomprising'successively treating suprarenal conadrenal cortexreplacement therapy in bilaterally ,50 tax tissue with each of threesolvents, the first or adrenalectomized animals and to maintain the saidsolvents being of the class consisting of ethyl lives of said animalsfor over 109 days following alcohoL -propyl alcohol, and acetone, thesecond adrenalectomy and to restore-.to normal condi-. of said solventsbeing of the class consisting of tions'anlmals prostrated by adrenalcortex defibenzol, sulphuric ether, petroleum ether and carciency. Y

a I r bon tetrachloride'and the third'of said solvents WILBUR WILL SSWINGLE.

I being acetone, 'separating the solution from the JbSEPH JOHN PFIFFNER.

